Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
  • C12P17/10—Nitrogen as only ring hetero atom

Definitions

• the present invention relates to a method for producing optically active 2-(N-substituted aminomethyl)-3-hydroxybutyric acid esters.
• Those compounds are useful, for example, as raw materials or intermediates for the synthesis of medicinal compounds required to be optically active.
• Optically active 2-(N-substituted aminomethyl)-3-hydroxybutyric acid esters are compounds important as intermediates for the synthesis of ⁇ -lactam antibiotics, typically thienamycin.
• a method known for the production of those compounds comprises stereospecifically and catalytically reducing the carbonyl group in position 3 of 2-(N-substituted aminomethyl)-3-oxobutyric acid esters by the hydrogenation reaction using a ruthenium-optically active phosphine complex (Non-Patent Document 1; Patent Document 1).
• this catalytic reduction method requires the use of a very expensive optically active phosphine ligand for attaining a high level of stereoselectivity and the use of a high hydrogen pressure of about 1 to 10 MPa; for these and other reasons, this method is not fully satisfactory from the commercial production and economical viewpoint.
• Patent Document 3 a compound having the (2S,3R) configuration was detected (Patent Document 3; Non-Patent Document 3).
• Patent Document 1 Japanese Kokai Publication Hei02-134349
• Patent Document 2 Japanese Kokai Publication Sho63-297360
• Patent Document 3 United States Patent Application Publication 2003/0139464
• Non-Patent Document 1 R. Noyori et al., “Stereoselective hydrogenation via dynamic kinetic resolution”, J. Am. Chem. Soc., 111, 9134 (1989)
• Non-Patent Document 2 Claudio Fuganti et al., “Microbial Generation of (2R,3S)- and (2S,3S)-Ethyl 2-Benzamidomethyl-3-hydroxybutyrate, a key intermediate in the synthesis of (3S,1′R)-3-(1′-hydroxyethyl)azetidin-2-one”, J. Chem, Soc. Perkin Trans., 1, (1993), 2247
• Non-Patent Document 3 Joo Hwan Cha et al., “Stereochemical control in diastereoselective reduction of ⁇ -substituted- ⁇ -ketoesters using a reductase purified from Kluyveromyces marxianus ”, Biotechnol. Lett., 24, 1695 (2002)
• the present inventors made investigations in an attempt to accomplish the above object and, as a result, discovered enzyme sources capable of stereoselectively reducing the carbonyl group of 2-(N-substituted aminomethyl)-3-oxobutyric acid esters to convert the same to the corresponding 2-(N-substituted aminomethyl)-3-hydroxybutyric acid esters having the (2S,3R) configuration and have now completed the present invention.
• the present invention relates to
• R 1 represents a lower alkyl group, which may optionally be substituted, an allyl group, an aryl group, which may optionally be substituted, or an aralkyl group, which may optionally be substituted and, as for R 2 and R 3
• R 3 is a hydrogen atom and R 2 represents a lower alkyl group, which may optionally be substituted, a lower alkoxy group, which may optionally be substituted, an aryl group, which may optionally be substituted, or an aralkyloxy group, which may optionally be substituted, or 2)
• R 3 and —COR 2 together represent a phthaloyl group
• R 1 , R 2 and R 3 are as defined above: is treated with an enzyme source capable of stereoselectively reducing said ester to the corresponding optically active 3-hydroxybutyric acid ester having the (2S,3R) configuration.
• the present invention provides a method for industrially producing 2-(N-substituted aminomethyl)-3-hydroxybutyric acid esters having the (2S,3R) configuration which are useful as intermediates for the production of medicinal compounds, among others.
• the 2-(N-substituted aminomethyl)-3-oxobutyric acid ester as an example of the substrate to be used in the reduction reaction in accordance with the invention is a compound represented by the general formula (6):
• R 1 represents a lower alkyl group, which may optionally be substituted, an allyl group, an aryl group, which may optionally be substituted, or an aralkyl group, which may optionally be substituted and, as for R 2 and R 3 ,
• R 3 is a hydrogen atom and R 2 represents a lower alkyl group, which may optionally be substituted, a lower alkoxy group, which may optionally be substituted, an aryl group, which may optionally be substituted, or an aralkyloxy group, which may optionally be substituted, or 2) R 3 and —COR 2 together represent a phthaloyl group.
• R 1 is as defined above and R 2 represents a lower alkyl group, which may optionally be substituted, a lower alkoxy group, which may optionally be substituted, an aryl group, which may optionally be substituted, or an aralkyloxy group, which may optionally be substituted
• R 3 and R 2 are related to each other in the manner mentioned above under 2
• the compound represented by the formula (6) is a compound represented by the formula (4):
• the term “lower” denotes that the relevant group contains 1 to 7 carbon atoms, preferably 1 to 4 carbon atoms.
• the lower alkyl group there may be mentioned, for example, a methyl group, an ethyl group, a chloromethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a tert-butyl group, an n-pentyl group and a cyclohexyl group, among others.
• Preferred are a methyl group, an ethyl group, an n-propyl group, a butyl group and the like. These groups may be substituted.
• the substituent or substituents are not particularly restricted but each may be selected from among halogen atoms, a hydroxyl group, an amino group, a nitro group, a cyano group and so forth, provided that they will not adversely affect the reduction reaction in the practice of the present invention.
• the aryl group which may optionally be substituted, is not particularly restricted but includes, for example, a phenyl group, an o-methylphenyl group, an m-methylphenyl group, a p-methylphenyl group, an o-methoxyphenyl group, an m-methoxyphenyl group, a p-methoxyphenyl group, an o-fluorophenyl group, an m-fluorophenyl group, a p-fluorophenyl group, an o-chlorophenyl group, an m-chlorophenyl group, a p-chlorophenyl group, an o-nitrophenyl group, an m-nitrophenyl group, a p-nitrophenyl group, an o-trifluoromethylphenyl group, an m-trifluoromethylphenyl group, a p-trifluoromethylphenyl group, a naphthyl
• the aralkyl group which may optionally be substituted, is not particularly restricted but may be a benzyl group, a p-hydroxybenzyl group or a p-methoxybenzyl group, for instance.
• the lower alkoxy group is not particularly restricted but includes a methyloxy group, an ethyloxy group, a chloromethyloxy group, an n-propyloxy group, an isopropyloxy group, an n-butyloxy group, an isobutyloxy group, a tert-butyloxy group, an n-pentyloxy group and a cyclohexyloxy group, among others.
• aralkyloxy group which may optionally be substituted, there may be mentioned a benzyloxy group, a p-hydroxybenzyloxy group and a p-methoxybenzyloxy group, among others; a benzyloxy group is preferred, however.
• R 1 is preferably an alkyl group containing 1 to 4 carbon atoms, more preferably a methyl group.
• R 2 is preferably a phenyl group, which may optionally be substituted, more preferably a phenyl group, a p-nitrophenyl group or a p-chlorophenyl group, still more preferably a phenyl group.
• R 3 and —COR 2 together form a phthaloyl group. Further, the case where R 1 is a methyl group and R 2 is a phenyl group is particularly preferred.
• the compound represented by the formula (6) given above is subjected to asymmetric reduction by treatment with an enzyme source capable of asymmetrically reducing said compound to give the corresponding optically active 2-(N-substituted aminomethyl)-3-hydroxybutylic acid ester represented by the general formula (5):
• Usable as the enzyme source in the practice of the invention is one derived from a microorganism capable of converting a 2-(N-substituted aminomethyl)-3-oxobutyric acid ester to the corresponding optically active 2-(N-substituted aminomethyl)-3-hydroxybutyric acid ester.
• the term “one derived from a microorganism” as used herein includes, within the meaning thereof, cells of the microorganism as such, a culture of the microorganism, a product obtained by subjecting cells of the microorganism to certain treatment, the enzyme obtained from the microorganism and, further, a transformant obtained by introduction of a DNA coding for the enzyme derived from the microorganism and having such reducing activity as mentioned above. These may be used singly or in combination of two or more species. These enzyme sources may be immobilized so that they may be used repeatedly.
• microorganism capable of converting 2-(N-substituted aminomethyl)-3-oxobutyric acid esters to optically active 2-(N-substituted aminomethyl)-3-hydroxybutyric acid esters can be found out by such a method as described below. Thus, for example, the following method is employed.
• a liquid medium having a composition comprising 40 g of glucose, 3 g of yeast extract, 6.5 g of diammonium hydrogen phosphate, 1 g of potassium dihydrogen phosphate, 0.8 g of magnesium sulf
• cells are collected by centrifugation, suspended in 0.5 to 5 ml of phosphate buffer containing 2 to 10% of glucose, added to 0.5 to 25 mg of methyl 2-benzamidomethyl-3-oxobutyrate and the like (belonging to the category of 2-(N-substituted aminomethyl)-3-oxobutyric acid esters) placed in advance in a test tube, and shake-cultured at 30° C. for 2 to 3 days. On that occasion, the cells collected by centrifugation may also be used after drying in a desiccator or using acetone.
• NAD + and/or NADP + may be added, together with glucose dehydrogenase and glucose or with formate dehydrogenase and formic acid.
• An organic solvent may be caused to coexist in the reaction system. After the conversion reaction, the reaction mixture is extracted with an appropriate organic solvent, and the 2-benzamidomethyl-3-hydroxybutyl esters formed are assayed by high-performance liquid chromatography, for instance.
• any of those microorganisms capable of converting the 2-(N-substituted aminomethyl)-3-oxobutyric acid esters to the corresponding (2S,3R)-2-(N-substituted aminomethyl)-3-hydroxybutyric acid esters can be used.
• microorganisms belonging to the genera Candida, Geotrichum, Galactomyces, Saccharomycopsis, Achromobacter, Arthrobacter, Bacillus, Brevundimonas, Xanthomonas, Devosia, Ralstonia, Lactobacillus, Leuconostoc, Microsporum and Moniliella , among others.
• Candida kefyr Candida oleophila, Candida maris, Geotrichum eriense, Galactomyces reessii, Saccharomycopsis malanga, Achromobacter xylosoxidans, Achromobacter denitrificans, Arthrobacter paraffineus, Arthrobacter nicotianae, Bacillus amylolyticus, Bacillus circulans, Bacillus cereus, Bacillus badius, Bacillus sphaericus, Brevundimonas diminuta, Xanthomonas sp., Devosia riboflavina, Ralstonia eutropha, Lactobacillus brevis, Lactobacillus helveticus, Leuconostoc pseudomesenteroides, Microsporum cookei and Moniliella acetoabatens , among others.
• microorganisms can generally be obtained from stock strains readily available or purchasable, although they may be isolated from the natural world. It is also possible to obtain microbial strains having properties favorable for the reaction in question by causing a mutation in these microorganisms.
• any of the media containing nutrient sources generally assimilable by these microorganisms can be used.
• a carbon source or carbon sources selected from among saccharides such as glucose, sucrose and maltose, organic acids such as lactic acid, acetic acid, citric acid and propionic acid, alcohols such as ethanol and glycerol, hydrocarbons such as paraffins, fats and oils such as soybean oil and rapeseed oil, and mixtures thereof; a nitrogen source or nitrogen sources selected from among ammonium sulfate, ammonium phosphate, urea, yeast extracts, meat extracts, peptone, corn steep liquor and so forth; and, further, a nutrient source or sources selected from among inorganic salts and vitamins, among others, as appropriately formulated and mixed up.
• An appropriate medium can be selected from among these depending on the microorganism employed.
• the cultivation of the microorganism can be carried out under ordinary conditions.
• the cultivation is preferably carried out aerobically within a pH range of 4.0 to 9.5 and a temperature range of 20° C. to 45° C. for 10 to 96 hours.
• the culture fluid containing cells of the microorganism in reacting the microorganism with a 2-benzamidomethyl-3-oxobutyric acid ester, the culture fluid containing cells of the microorganism can be submitted as such to the reaction, and the culture broth can also be used in the form of a concentrate.
• cells or a cell treatment product obtained by subjecting the culture fluid to such treatment as centrifugation can also be used.
• the cell treatment product derived from the microorganism is not particularly restricted but includes, for example, dried cells obtained by dehydration treatment using acetone or diphosphorus pentaoxide or utilizing a desiccator or electric fan, surfactant treatment products, lytic enzyme treatment products, immobilized cells and cell-free extracts obtained by cell disruption, among others. Further, an enzyme catalyzing the reduction reaction stereoselectively as purified from the culture may also be used.
• the substrate 2-(N-substituted aminomethyl)-3-oxobutyric acid esters may be added all at once at the beginning of the reaction or may be added in divided portions according to the progress of the reaction.
• the reaction temperature is generally 10 to 60° C., preferably 20 to 40° C., and the pH during the reaction is within the range of 2.5 to 9, preferably 5 to 9.
• the amount of the enzyme source in the reaction mixture can be appropriately determined according to the capability thereof to reduce the substrate.
• the substrate concentration in the reaction mixture is preferably 0.01 to 50% (w/v), more preferably 0.1 to 30% (w/v).
• the reaction is generally carried out with shaking or with stirring under aeration.
• the reaction time is properly determined according to the substrate concentration, enzyme source amount and other reaction conditions. Generally, the various conditions are preferably selected so that the reaction may be completed in 2 to 168 hours.
• such an energy source as glucose, ethanol or isopropanol is preferably added in a proportion of 0.5 to 30% so that favorable results may be obtained. It is also possible to promote the reaction by adding such a coenzyme generally required in carrying out a biological reduction reaction as reduced nicotinamide adenine dinucleotide (hereinafter referred to as NADH for short) or reduced nicotinamide adenine dinucleotide phosphate (hereinafter referred to as NADPH for short). In this case, more specifically, they are directly added to the reaction mixture.
• NADH reduced nicotinamide adenine dinucleotide
• NADPH reduced nicotinamide adenine dinucleotide phosphate
• the reaction is preferably carried out in the simultaneous presence of an enzyme reducing NAD + or NADP + to the respective reduced form as well as a substrate for the reduction to give favorable results.
• an enzyme reducing NAD + or NADP + to the respective reduced form as well as a substrate for the reduction to give favorable results.
• glucose dehydrogenase is caused to coexist as the enzyme for reduction to the reduced form and glucose as the substrate for reduction
• formate dehydrogenase is caused to coexist as the enzyme for reduction to the reduced from and formic acid as the substrate for reduction.
• optically active 2-(N-substituted aminomethyl)-3-hydroxybutyric acid esters in the same manner by using, in lieu of the enzyme (reductase) catalyzing the reduction reaction according the present invention, a transformant harboring a DNA coding for that enzyme.
• the optically active 2-(N-substituted aminomethyl)-3-hydroxybutyric acid ester in the same manner by using a transformant harboring both a DNA coding for the reductase according to the present invention and a DNA coding for a polypeptide capable of catalyzing coenzyme reproduction.
• a transformant harboring both a DNA coding for the reductase according to the invention and a DNA coding for a polypeptide capable of catalyzing coenzyme reproduction is used, the optically active 3-hydroxybutyric acid esters can be produced more efficiently without the need of separately preparing and adding the enzyme for coenzyme reproduction.
• the transformant harboring a DNA coding for the polypeptide according to the invention or the transformant harboring both a DNA coding for the polypeptide according to the invention and a DNA coding for a polypeptide capable of catalyzing coenzyme reproduction can be used in the production of the optically active 3-hydroxybutyric acid esters.
• the treatment product derived from the transformant, so referred to herein, is as described hereinabove.
• the transformant harboring both a DNA coding for the reductase according to the invention and a DNA coding for a polypeptide capable of catalyzing coenzyme reproduction can be obtained by integrating both a DNA coding for the reductase according to the invention and a DNA coding for a polypeptide capable of catalyzing coenzyme reproduction into one and the same vector and introducing the resulting recombinant vector into host cells or by integrating these two DNAs respectively into two vectors belonging to different incompatibility groups and introducing the two recombinant vectors into host cells.
• pNTDRG1 obtainable by introduction of the Bacillus megaterium -derived glucose dehydrogenase gene into the expression vector pNTDR described in WO 2004/027055.
• E. coli HB101 obtainable by transforming E. coli HB101 with the vector mentioned above.
• the cultivation of the transformant harboring a DNA coding for the reductase according to the invention and the cultivation of the transformant harboring both a DNA coding for the reductase according to the invention and a DNA coding for a polypeptide capable of catalyzing coenzyme reproduction can be carried out using ordinary liquid nutrient media containing a carbon source(s), a nitrogen source(s), an inorganic salt(s) and an organic nutrient(s), among others, so long as the transformant can grow therein.
• a surfactant as Triton (product of Nakalai Tesque, Inc.), Span (product of KANTO CHEMICAL CO., INC.) or Tween (product of Nakalai Tesque, Inc.).
• a surfactant such as Triton (product of Nakalai Tesque, Inc.), Span (product of KANTO CHEMICAL CO., INC.) or Tween (product of Nakalai Tesque, Inc.).
• an organic solvent insoluble in water as ethyl acetate, butyl acetate, isopropyl ether, toluene or hexane may be added to the reaction mixture. It is further possible to add such an organic solvent soluble in water as methanol, ethanol, acetone, tetrahydrofuran or dimethyl sulfoxide for the purpose of increasing the solubility of the substrate.
• the method for recovering the optically active 2-(N-substituted aminomethyl)-3-hydroxybutyric acid esters formed by the reduction reaction is not particularly restricted but the optically active 2-(N-substituted aminomethyl)-3-hydroxybutyric acid esters can be readily obtained in a highly pure form by extracting the same directly from the reaction mixture or from cells or the like separated therefrom with such a solvent as ethyl acetate, toluene, tert-butyl methyl ether or hexane and, after dehydration, purifying the same by distillation or silica gel column chromatography, for instance.
• a solvent as ethyl acetate, toluene, tert-butyl methyl ether or hexane
• methyl 2-benzamidomethyl-3-oxobutyrate (Examples 1 to 13), tert-butyl 2-benzamidomethyl-3-oxobutyrate (Examples 14 to 16), methyl 2-acetamidomethyl-3-oxobutyrate (Examples 17 and 18) and methyl 2-phthaloylamidomethyl-3-oxobutyrate (Examples 19 and 20) were used as examples of the reduction reaction substrate 2-(N-substituted aminomethyl)-3-oxobutyric acid esters.
• the reaction using a transformant is shown in Example 21.
• the yield in the reduction reaction and the optical purity of the product were determined using the specified microorganism, enzyme, or enzyme and coenzyme regeneration system enzyme, among others. According to the measurement results, it was found, in each example, that the 3-hydroxybutyric acid esters having the (2S,3R) configuration can be produced with high efficiency.
• a liquid medium (pH 7) having a composition comprising 40 g of glucose, 3 g of yeast extract, 6.5 g of diammonium hydrogen phosphate, 1 g of potassium dihydrogen phosphate, 0.8 g of magnesium sulfate heptahydrate, 60 mg of zinc sulfate heptahydrate, 90 mg of iron sulfate heptahydrate, 5 mg of copper sulfate pentahydrate, 10 mg of manganese sulfate tetrahydrate and 100 mg of sodium chloride (each per liter) was distributed in 5-ml portions into large-sized test tubes, and steam-sterilized at 120° C. for 20 minutes.
• This cell suspension was added to a test tube containing 2.5 mg of methyl 2-benzamidomethyl-3-oxobutyrate placed therein in advance and the reaction was allowed to proceed at 30° C. for 24 hours. Thereafter, 1 ml of ethyl acetate was added to each reaction mixture, followed by thorough mixing. A portion of the organic layer was analyzed using a HPLC equipped with Daicel Chemical Industries' Chiralpak AD-H (250 mm ⁇ 4.6 mm), and the yield and optical purity of the reaction product were determined. The results thus obtained are summarized in Table 1.
• a liquid medium (pH 7) having a composition comprising 10 g of meat extract, 10 g of peptone, 5 g of yeast extract and 3 g of sodium chloride (each per liter) was distributed in 7-ml portions into large-sized test tubes and steam-sterilized at 120° C. for 20 minutes. These liquid media were respectively inoculated aseptically with the microorganisms shown below in Table 2 (the inoculum size being one loopful), followed by 72 hours of shake culture at 30° C. After cultivation, each culture fluid was centrifuged and the thus-collected cells were suspended in 0.5 ml of 100 mM phosphate buffer (pH 6.5) containing 1% of glucose.
• This cell suspension was added to a test tube containing 2.5 mg of methyl 2-benzamidomethyl-3-oxobutyrate placed therein in advance and the reaction was allowed to proceed at 30° C. for 24 hours. Thereafter, 1 ml of ethyl acetate was added to each reaction mixture and, after thorough mixing, a portion of the organic layer was analyzed under the analysis conditions described in Example 1 and the yield and optical purity of the product were determined. The results thus obtained are summarized in Table 2.
• a liquid medium (pH 7) having a composition comprising 10 g of glucose, 10 g of peptone, 10 g of meat extract, 5 g of yeast extract 1 g of sodium chloride and 0.5 g of magnesium sulfate heptahydrate (each per liter) was distributed in 5-ml portions into large-sized test tubes and steam-sterilized at 120° C. for 20 minutes.
• These liquid media were respectively inoculated aseptically with the microorganisms shown below in Table 3 (the inoculum size being one loopful), followed by 72 hours of shake culture at 28° C. After cultivation, each culture fluid was centrifuged and the thus-collected cells were suspended in 1 ml of 100 mM phosphate buffer (pH 6.5) containing 1% of glucose.
• This cell suspension was added to a test tube containing 1 mg of methyl 2-benzamidomethyl-3-oxobutyrate placed therein in advance and the reaction was allowed to proceed at 30° C. for 24 hours. Thereafter, 2 ml of ethyl acetate was added to each reaction mixture and, after thorough mixing, a portion of the organic layer was analyzed under the analysis conditions described in Example 1 and the yield and optical purity of the product were determined. The results thus obtained are summarized in Table 3.
• MSR medium product of Difco Laboratories
• This cell suspension was added to a test tube containing 1 mg of methyl 2-benzamidomethyl-3-oxobutyrate placed therein in advance and the reaction was allowed to proceed at 30° C. for 24 hours. Thereafter, 2 ml of ethyl acetate was added to each reaction mixture, followed by thorough mixing. A portion of the organic layer was analyzed under the analysis conditions described in Example 1 and the yield and optical purity of the product were determined. The results thus obtained are summarized in Table 4.
• reaction mixture was extracted with three 45-ml portions of ethyl acetate, and the organic layers obtained were combined and dried over anhydrous sodium sulfate.
• the sodium sulfate was filtered off, the organic solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography to give 4.4 g of methyl (2S,3R)-2-benzamidomethyl-3-hydroxybutanoate.
• This product had an optical purity of 99% ee or above, and the diastereoselectivity was 89.8% de.
• a liquid medium (pH 7) having a composition comprising 10 g of meat extract, 10 g of peptone, 5 g of yeast extract and 3 g of sodium chloride (each per liter) was distributed in 7-ml portions into large-sized test tubes and steam-sterilized at 120° C. for 20 minutes. These liquid media were respectively inoculated aseptically with the microorganisms shown below in Table 5 (the inoculum size being one loopful), followed by 72 hours of shake culture at 30° C. Thereafter, each culture fluid was centrifuged and the thus-collected cells were suspended in 0.5 ml of 100 mM phosphate buffer (pH 6.5) containing 1% of glucose.
• This cell suspension was added to a test tube containing 0.5 mg of ethyl 2-benzamidomethyl-3-oxobutyrate placed therein in advance and the reaction was allowed to proceed at 30° C. for 24 hours. Thereafter, 1 ml of ethyl acetate was added to each reaction mixture and, after thorough mixing, a portion of the organic layer was analyzed using a HPLC equipped with Daicel Chemical Industries' Chiralpak AD-H (250 mm ⁇ 4.6 mm), and the yield and optical purity of the reaction product were determined. The results thus obtained are summarized in Table 5.
• a liquid medium (pH 7) having a composition comprising 10 g of meat extract, 10 g of peptone, 5 g of yeast extract and 3 g of sodium chloride (each per liter) was distributed in 7-ml portions into large-sized test tubes and steam-sterilized at 120° C. for 20 minutes. These liquid media were respectively inoculated aseptically with the microorganisms shown below in Table 6 (the inoculum size being one loopful), followed by 72 hours of shake culture at 30° C. Thereafter, each culture fluid was centrifuged and the thus-collected cells were suspended in 0.5 ml of 100 mM phosphate buffer (pH 6.5) containing 1% of glucose.
• This cell suspension was added to a test tube containing 0.5 mg of tert-butyl 2-benzamidomethyl-3-oxobutyrate placed therein in advance and the reaction was allowed to proceed at 30° C. for 24 hours. Thereafter, 1 ml of ethyl acetate was added to each reaction mixture and, after thorough mixing, a portion of the organic layer was analyzed using a HPLC equipped with Daicel Chemical Industries' Chiralpak AD-H (250 mm ⁇ 4.6 mm), and the yield and optical purity of the reaction product were determined. The results thus obtained are summarized in Table 6.
• E. coli HB101 (pNTDRG1) (FERM BP-08458; cf. WO 2004/027055) was cultured in 2 ⁇ YT medium containing 120 ⁇ g/ml of ampicillin.
• 2 ⁇ YT medium containing 120 ⁇ g/ml of ampicillin.
• To 30 ml of the thus-obtained culture fluid were added 2 g of glucose, 50 mg of NAD and 3 g of methyl 2-benzamidomethyl-3-oxobutyrate, and the mixture was stirred at 30° C. while the pH of the reaction mixture was maintained at 6.5 with 6 N NaOH. After 24 hours of reaction, the reaction mixture was extracted with three 30-ml portions of ethyl acetate, and the organic layers obtained were combined and dried over anhydrous sodium sulfate.

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